Naslov
Human keratinocyte culture in vitro

Autori
Stanović, Silvana ; Martin-Kleiner, Irena ; Kušec, Rajko ; Tudorić-Ghemo, Ivana ; Jakić-Razumović, Jasminka ; Kljenak, Antun ; Fattorini, Ivan ; Boranić, Milivoj

Vrsta, podvrsta i kategorija rada
Radovi u zbornicima skupova, cjeloviti rad (in extenso), znanstveni

Izvornik
Current Studies in Biotechnology, Volume I - Biomedicine / Kniewald, Zlatko et al. - Zagreb : Croatian Society for Biotechnology, 2000, 75-83

Skup
Scientific Conference Biotechnology and Biomedicine, with international participation

Mjesto i datum
Zagreb, Hrvatska, 22.02.1999. - 23.02.1999

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Domaća recenzija

Ključne riječi
keratinocytes; cell culture; cell proliferation; defined keratinocyte serum-free medium (DK-SFM); CD10/NEP

Sažetak
Human epidermal cells, keratinocytes, can be cultured in vitro using a selective defined keratinocyte serum-free medium (DKSFM) or a feeder-layer of irradiated murine fibroblasts. We describe our experience with keratinocyte cultures in DKSFM. Keratinocytes were isolated from human foreskins obtained at routine pediatric circumcisions. Epidermis was separated from dermis by enzymatic digestion in dispase and dissolved into single cell suspensions by trypsin. Keratinocyte suspensions in DKSFM were seeded into 25 cm2 tissue culture flasks or into 35 mm, 60 mm or 100 mm Petri dishes and maintained at 37 °C, 5% CO2 and maximal humidity. The medium was renewed every 2 days until the cells reached confluence. In successful cultures that occurred 2-3 weeks after the initiation. A satisfactory growth was obtained with one-third (35%) of the isolates. The failures were due to contamination (32%), inappropriate plastics (13%) or for reasons not sufficiently clear (19%). Primary cultures that reached confluence were trypsinized and the cells frozen in liquid nitrogen or re-seeded into secondary cultures for expansion. Samples of primary or secondary keratinocytes were cytospinned for immunocytochemical examination. The keratinocyte proliferation rate was measured in a microplate assay using a colorimetric method. DNA extracted from cultured keratinocytes showed by PCR the presence of the gene coding the membrane metallopeptidase CD10 (EC 3.4.24.11). Thus, primary cultures of human keratinocytes in DKSFM can be a convenient experimental model provided a satisfactory growth is obtained.

Izvorni jezik
Engleski

Znanstvena područja
Kliničke medicinske znanosti

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